o r i g i n a l r es e a r c h
Analysis of a Complex Polyploid Plant Genome
using Molecular Markers: Strong Evidence
for Segmental Allooctoploidy in Garden Dahlias
Stephan Schie, Rajiv Chaudhary, and Thomas Debener*
In some plant genera that contain species with complex genomes,
the level and type of ploidy are still unknown due to a lack of
characterized reference species and contradictory results from
genetic and cytogenetic studies. Herein, we present the analysis
of the genome of garden dahlias using molecular markers; this
species is one for which the genome ploidy has remained controversial. We generated simple-sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) data from two segregating populations of garden dahlias. The combined analysis of SSR
marker segregation, the ratio of single-dose to multidose markers,
the ratio of markers linked in coupling and repulsion, and map
construction revealed a predominantly autooctoploid genome with
a low degree of preferential pairing. This finding indicates that
dahlias are segmental allooctoploids that originated from autotetraploid ancestral genomes. Our results demonstrate that marker
analysis is a suitable method for ploidy analysis in nonmodel
crops. Novel marker techniques, such as restriction site associated
DNA, will make this analysis even more effective before whole
genome sequencing can be realized for these crops.
Published in The Plant Genome 7
doi: 10.3835/plantgenome2014.01.0002
© Crop Science Society of America
5585 Guilford Rd., Madison, WI 53711 USA
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the pl ant genome
P
olyploidy, the situation in which genomes are com-
Abstract
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november 2014
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vol . 7, no . 3 prised of more than two homologous sets of chromosomes, is a widespread phenomenon among higher
plants and a major factor shaping the structure and evolution of plant genomes (Adams and Wendel, 2005). It is
estimated that 20 to 70% of all species of higher plants
are polyploids, but experimental evidence is lacking for
most species (Otto and Whitton, 2000; Bennett, 2004).
Two principal types of polyploids can be distinguished in
angiosperms. Autopolyploidy results from the fusion of
unreduced gametes within a species. Thus, the chromosomes pair randomly and form multivalents (more than
two chromosomes form synaptic pairs during meiosis),
and polysomic segregation (chromosomes are randomly
distributed among the gametes) of the loci occurs. In contrast, allopolyploids are the product of the fusion between
unreduced gametes from different species. As a result,
chromosome pairing among the subgenomes is preferential, and a high number of bivalents (only two chromosomes pair) are observed during meiosis (Stebbins, 1947).
Furthermore, there is little or no intergenomic recombination among the subgenomes; thus, the subgenomes
are preserved, and the inheritance of the loci is usually
disomic (e.g., they behave as diploids for each particular
subgenome). However, in addition to the two main forms
of ploidy, various intermediate types in which different
regions of the genome display varying degrees of preferential chromosome pairing have been observed (Stebbins, 1947; Bennett, 2004). These so-called “segmental
S. Schie and T. Debener, Leibniz Universitaet Hannover, Institute
for Plant Genetics, Molecular Plant Breeding Herrenhaeuser Str.
2, 30419 Hannover, Germany; R. Chaudhary, Indian Institute of
Vegetable Research, Post Bag No. 01; P. O. Jakhini (Shahanshapur)
Varanasi- 221 305 Uttar Pradesh, India. Received 6 Jan. 2014.
*Corresponding author ([email protected]).
Abbreviations: AFLP, amplified fragment length polymorphism; CP,
cross pollination mode; SSR, simple-sequence repeat.
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allopolyploids” usually display a mixed disomic-polysomic inheritance. As a number of plant genomes have
been sequenced, it has become apparent that almost all
functional diploid species have doubled their genomes
at least once in their evolutionary history. These events
were followed by a subsequent reduction in genome size
and complexity (diploidization) that reduced the genome
again to a diploid state but left numerous duplicated chromosomal fragments (Song et al., 1995; Wolfe, 2001; Kashkush et al., 2002). Even plants with small and compact
genomes, such as Arabidopsis, still show extended regions
that originated from ancient duplication events (The Arabidopsis Genome Initiative, 2000).
Dahlias are popular ornamental plants that are
mainly used as bedding and pot plants or as cut flowers.
The genus Dahlia contains 38 species, most of which have
either 32 or 64 chromosomes. Garden dahlias exclusively
contain genotypes with 64 chromosomes. The genus
originated in Mexico and Central America and was introduced to Europe around 1790, when the first seeds were
brought to the Madrid botanical garden (Cavanilles, 1791;
Sørensen, 1969), followed by further imports of the seeds
and whole plants of wild species and species hybrids.
Intercrosses between these genotypes led to hybrids that
are now classified as Dahlia variabilis (Desfontaines, 1829;
Hansen and Hjerting, 1996). The observation that some
wild Dahlia species are closely related to D. variabilis,
which have 2n = 32 chromosomes, led to the conclusion that D. variabilis is a polyploid species. However,
the ploidy level of both the wild and cultivated species
remains unclear, as Sørensen assumed that the wild species with 2n = 32 were already tetraploids, thus classifying
D. variabilis as an octoploid (Sørensen, 1969). Attempts
to solve this problem have led to contrasting results. As
an example, meiotic chromosome pairing was found to
occur mainly in bivalents, which is expected of allopolyploids (Lawrence, 1929; Gatt et al., 1998). In contrast,
studies concerning the inheritance of flower pigments
found polysomic segregation patterns, which are typical
of autopolyploids (Lawrence and Scott-Moncrieff, 1935;
Lawrence, 1970). However, cytological methods often
do not lead to conclusive results concerning the type of
ploidy (Sybenga, 1996; Benavente and Sybenga, 2004).
Here, recent molecular methods, such as the ratio of single-dose to multidose molecular markers (Da Silva et al.,
1993) and the ratio of markers linked in coupling to the
markers linked in repulsion (Wu et al., 1992), have led to a
more precise characterization. Here, a single-dose marker
is defined as a marker allele in only one copy (e.g., Aaaa
in a tetraploid) whereas a multidose marker is defined
as a marker allele with more than one copy (for example
AAaa, AAAa, or AAAA in a tetraploid).
A single-dose marker present in only one parent (uniparental marker) has a theoretical segregation
ratio of 1:1 (presence:absence) in an F1 progeny of both
autopolyploids and allopolyploids. Likewise, biparental
markers will segregate in a 3:1 (presence:absence) ratio
in both auto- and allopolyploids. In contrast, multidose
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markers have more complex segregation ratios that differ
between autopolyploids and allopolyploids. The expected
ratios for singledose to multidose markers is 0.56:0.44
in allopolyploids and 0.7:0.3 in autopolyploids (Da Silva
et al., 1993) so that the type of ploidy can be inferred if a
larger number of markers is tested for singledose versus
multidose segregation.
Concerning the ratio of markers in coupling and
repulsion, differences in occurrence among autopolyploids and allopolyploids can be exemplified by considering a tetraploid case for an uniparental locus with four
different alleles: In an autotetraploid, the alleles A1, A2,
A3, and A4 would be distributed to the gametes in all
six possible pairwise combinations (A1A2, A1A3, A1A4,
A2A3, A2A4, A3A4). If two dominant marker fragments are closely linked to locus A, but to two different
alleles (A1 and A2), their linkage is difficult to detect in
small populations because no clear repulsion situation
occurs and the distribution would not be sufficiently
different from ratios expected for independent segregation. In contrast, an allotetraploid genome with four
different alleles organized in two separate subgenomes
(A1A2B1B2) leads to gametes of the type A1B1, A1B2,
A2B1, or A2B2, but not A1 and A2 or B1and B2 within
the same gametes (except in the rare cases of double
reduction at meiosis). Here two different markers linked
to either allele A1 or to allele A2 (or likewise B1 and B2)
would be easily found to be linked in repulsion as they
would not occur within the same gamete. As the second
parent might be identical for some of the alleles, this
analysis also needs larger numbers of markers to find
a sufficient number of cases where distinct alleles only
occur in one of the parental genotypes.
Sequencing individual genes is another method that
could reveal both the copy number of the genomes and
uncover the potential differentiation of the subgenomes.
However, due to the widespread presence of duplicated
genomic regions in most plant genomes, the differentiation
between the allelic copies of orthologous genes and paralogous genes is a major challenge without additional information concerning the relatedness of the subgenomes.
Aims of the Current Work
Whole genome sequencing of Dahlia variabilis was
beyond our reach due to the large genome size, approximately 9 pg per 2 C, for most dahlia varieties, which corresponds to more than 8800 Mb (Temsch et al., 2008; C
values are the genome size of the haploid genome of an
organism). Therefore, we used molecular markers to gain
information on the extent and type of polyploidy present
in the Dahlia genus. This is an important prerequisite for
the development of superior breeding strategies, as it will
determine to what extent single Mendelian traits can be
selected in Dahlia progenies and to what extent molecular
markers can aid in the selection of single genes and QTLs.
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Materials and Methods
Plant Material
The plant material was extracted from two segregating
populations. Population K1, which contained 130 individuals, resulted from a cross between a pot dahlia (T8, genotype collection of Leibniz Universitaet Hannover) and a
garden dahlia (M331-6, collection of M. Otto, Lueneburg,
Germany). Population K1 was generated by the hand pollination of isolated individuals. As T8 had little viable pollen, single flowers were not emasculated before pollination.
Population K5, which contained 173 progeny, resulted
from a cross between two garden dahlias (Karneol as the
maternal and Nordlicht as the paternal parents). Both
parents were part of the genotype collection of M. Otto
(Lueneburg, Germany). Population K5 was generated by
co-cultivation of both parents in isolated field plots. The
few cases of self-pollination of Karneol and any unwanted
pollination events by other genotypes were later excluded
by analyses with molecular markers.
Analysis of the Molecular Markers
Table 1. Maximum number of simple-sequence repeat
(SSR) alleles per genotype in the Dahlia populations K1
and K5.
Number of maximum alleles per genotype
SSR Marker
Population K1
Population K5
DV01
DV02
DV03
DV04
DV05
DV06
DV07
DV08
DV09
DV10
DV11
DV12
DV13
HT292
2
3
6
2
6
3
3
7
4
8
5
4
7
4
2
3
5
2
5
3
4
7
4
6
6
4
7
4
Genomic DNA was extracted from 65 mg of dried leaves
using the DNeasy Plant Maxi Kit (Qiagen, Hilden, Germany), as described in Schie and Debener (2013). The
SSR markers DV01, DV02, and DV03 were previously
described in Schie and Debener (2013).
Additional SSR markers were generated by 454
sequencing of polymerase chain reaction products from
15 random amplified polymorphic DNA markers that
were generated based on the Karneol genotype. A total of
11 repetitive SSR motifs were detected by analyzing the
sequences with Tandem Repeat Finder (Benson, 1999)
using the alignment parameters 2, 7, 7, a minimum alignment score of 30, and a maximum period size of 50. Primers were generated using an internet-based version of the
Primer 3 software using standard settings (http://primer3.
sourceforge.net/, verified 27 June 2014; Rozen and Skaletsky, 2000). The SSR marker HT292 is a marker from
Helianthus annuus and was described by Heesacker et al.
(2008). The AFLP markers were generated on genomic
DNA of the K5 population, as described earlier (Wegner
and Debener, 2008), using HindIII and MseI as restriction
enzymes and four selective bases for the final amplification.
Results
Statistical Analysis
As allo- and autopolyploids display different ratios of
single-dose to multidose markers, we analyzed a total of
1334 segregating AFLP markers and SSR fragments with
a dominant scoring scheme in the K5 population. Singledose markers represent a single dominant segregating
locus in the simplex configuration, whereas multidose
markers represent more than one dominant allele, from
either one or both of the parents. The distinction between
single and multidose markers was determined by calculating the geometric mean of the theoretically expected
distributions for a given number of progeny; each marker
fragment was then assigned to one of the two classes. The
geometric mean is a commonly applied method used to
Basic statistical calculations were conducted in Excel
2003 (Microsoft Corp., Redmond, WA). Linkage analysis
and map construction were performed in Joinmap 4.0
(Van Ooijen, 2006) using the Kosambi mapping function
(Kosambi, 1944; grouping LOD = 6; mapping LOD = 6).
schi e et al .: dahlia ploi dy Analysis of the SSR Allele Number
and Segregation
The two segregating populations, K1 and K5, were analyzed using 14 SSR markers. For tetraploids, a maximum
of four different alleles would be expected for each locus,
whereas a maximum of eight markers would be expected
for octoploid individuals. The number of different alleles
per genotype ranged from two to a maximum of eight in
the K1 population and from two to a maximum of seven in
the K5 population (Table 1). In both populations, six markers displayed more than four fragments, which would be
expected for a single-dose marker in a tetraploid organism.
Furthermore, allopolyploids and autopolyploids differ in the segregation patterns of their SSR alleles, as the
alleles from the identical diploid subgenomes will not be
inherited together in allopolyploids. The segregation patterns of the SSR markers in both populations showed a
free combination of all SSR alleles, such that any division
into diploid subgenomes can be excluded.
Analysis of Single-Dose vs. Multidose Markers
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Table 2. Ratio of single-dose to multidose markers in
the K5 population compared with the expected values
for allo- and autopolyploid populations.
Single-dose markers
Multidose markers
Markers total
Ratio SD:MD†
Chi square value
Observed
Allopolyploid
Autopolyploid
931
403
1334
–
–
747.04
586.96
1334
0.56:0.44
102.96*
933.8
400.2
1334
0.7:0.3
0.028 ns‡
* Statistically significant at the 0.05 probability level.
†
Ratio of single-dose markers to multidose markers.
‡
ns, not significant.
distinguish between single-dose and multidose markers;
this method defines the mean between the expected segregation ratios for markers with only one dominant allele
and for markers with more than one dominant allele.
Therefore, it allows us to assign each marker to one of the
two classes (Mather, 1957; Grivet et al., 1996). In contrast
with the c2 test, this method allows for the assignment of
skewed markers to one of the two classes. The segregation
thresholds were 1.73 for uniparental markers (marker
allele present in only one of the parents) and 6.71 for biparental markers (marker allele present in both parents);
thus, each uniparental marker with less than a 1.73 ratio
and biparental markers with ratios < 6.71 were classified as single-dose markers. All markers exceeding these
threshold ratios were classified as multidose markers.
As a result, we observed 931 single-dose markers
and 403 multidose markers (Table 2). The deviation from
the expected ratio for autopolyploids was not significant,
whereas a highly significant deviation from the expected
values occurred for the allopolyploids.
Analysis of the Ratio of Coupling vs.
Repulsion Linkages
Using only the uniparental single-dose AFLP markers in
a subset of 75 progenies from the K5 population, we performed linkage analysis by generating a copy of the dataset in which each marker position was inverted (presence
changed to absence and vice versa), and the inverted copy
was joined to the original dataset as described earlier
(Wu et al., 1992). This dataset was then analyzed using
the Joinmap mapping software (v. 4.0, Van Ooijen, 2006)
in BC1 mode (backcross mode), and the markers were
grouped based on the independence LOD values, with a
maximal recombination frequency of 0.35. The inverted
dataset was checked for linkages to the original dataset.
Linkages within the original dataset were classified as
coupling linkages. Linkages between the inverted and
the original dataset were classified as repulsion linkages.
For this analysis, we used the 569 uniparental single-dose
markers for both parents of the K5 population (284 from
parent Karneol and 285 from parent Nordlicht). Of the
569 markers, 402 markers were linked in coupling, but
no marker was linked in repulsion. The remaining 167
markers were not found to be linked to any other marker.
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Table 3. Single-dose markers observed in coupling
and repulsion in the segregating progeny of the K5
population with respect to the expected values for the
different types of polyploidy.
Observed Allopolyploid
Markers linked in coupling
Markers linked in repulsion
Markers total
Ratio coupling:repulsion§
Chi square value
402
0
402
–
–
201
201
402
1:1
402.00*
Autopolyploid Autopolyploid
(>4x)‡
(4x)†
321.6
80.4
402
1:0.25
100.50*
402
0
402
1:0
0.00 ns¶
* Statistically significant at the 0.05 probability level.
†
Tetraploid.
‡
Ploidy level higher than tetraploid.
§
Ratio of markers linked in the coupling phase to markers linked in repulsion phase.
¶
ns, not significant.
Compared with the ratios of markers in coupling and
repulsion expected for allopolyploids, autotetraploids and
autopolyploids with higher ploidy levels, our observation
only fits the expected ratio for autopolyploids with ploidy
levels higher than tetraploid (Table 3).
Map Construction
For map construction, a total of 1293 markers were used,
and biparental single-dose markers were also included
in this analysis. In contrast with the method described
above, maps were constructed in the cross pollination
(CP, two heterozygous genotypes intercrossed) mode with
the same linkage thresholds of the LOD values from 6.0
to 10.0. In total, 151 linkage groups, 73 for Karneol and
78 for Nordlicht, were calculated. In contrast to the determination of linkage in the BC1 mode described above,
map construction in the CP mode revealed two markers
that were linked in repulsion. Markers displayed a high
degree of skewed segregation, with 55.3% derived from
Karneol and 40.7% from Nordlicht. The total map length
was 2571 cM for Karneol and 3086 cM for Nordlicht.
Given that both genotypes have a chromosome number of
64, the number of linkage groups exceeded this number.
However, for a polyploid genome with 64 chromosomes,
the total number of markers was far too low to reach saturation of the map. Therefore, a match between the number of linkage groups and the number of chromosomes
cannot be expected. Homologous linkage groups were
identified by including 14 SSR markers and 170 biparental
AFLP markers. From a total of 151 linkage groups, 103
could be assigned to 29 different homologous groups. Up
to four different SSR alleles linked the individual linkage
groups to the homologous groups (Table 4).
Discussion
Degree of Ploidy
The methods of choice for determining the degree of
ploidy in plants are either cytogenetic analysis of the
number of mitotic chromosomes or flow cytometry of
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Table 4. Number of simple-sequence repeat (SSR) alleles mapped to homologous linkage groups derived
from parents of the K5 population.
Homologous
group Number
Number of individual
linkage groups†
1
7(K), 8(N)
2
3
4(K), 9(N)
4(K), 8(N)
4
5
6
7
3(K), 3(N)
2(K), 1(N)
2(K), 1(N)
3(N)
SSR marker
Alleles
Karneol
Alleles
Nordlicht
DV13
DV10
DV03
DV05
DV07
DV12
HT292
DV08
DV02
DV09
DV11
3
2
2
2
1
1
1
3
2
2
0
4
2
4
3
2
2
1
1
1
0
3
Number of calculated linkage groups for the female parent Karneol (K) and the male parent
Nordlicht (N) within each homologous group.
†
crude nuclei preparations stained with a fluorescent DNA
binding dye. However, both methods require properly
characterized control samples. In the case of dahlias, these
samples are not available, as there has been no agreement
on the basic chromosome number in this genus.
Therefore, we utilized molecular markers and a combination of strategies to analyze the ploidy level of the
genus Dahlia. The number of SSR alleles per genotype
exceeded four alleles in six out of the 14 markers tested,
indicating a ploidy level higher than 4x. As the number
of chromosomes (64) cannot be divided by six to obtain
a natural number, an octoploid set of chromosomes must
be assumed for the 2n = 64 chromosome varieties. This
finding is supported by the ratio of the markers linked
in coupling to those linked in repulsion. The fact that
no markers were linked in repulsion according to the
BC1 mode of Joinmap is a clear indication that dahlias
are polyploids with a ploidy level higher than tetraploid,
which is in agreement with the SSR data. Thus, D. variabilis can be concluded to be an octoploid with a basic chromosome set of x = 8. Because we used next generation
sequencing data to generate some of the SSR markers, we
also tried to align sequence reads to analyze the maximal
number of alleles for the individual contigs. However,
due to limited sequence information and problems distinguishing alleles from paralogous sequences, conclusive
results could not be obtained (data not shown).
Type of Ploidy
The segregation pattern of SSR markers also provided
information about the type of ploidy. The observation
that the fragments of every SSR marker were inherited
in all combinations in the segregating progeny indicated
a predominantly autotetraploid mode of inheritance.
Co-segregation, in agreement with the presence of subgenomes as expected for allopolyploids, was not observed.
This conclusion was further supported by the ratio of
AFLP markers linked in coupling versus those linked in
schi e et al .: dahlia ploi dy repulsion. The observed ratio of 1:0 for coupling versus
repulsion markers contrasts with the expected ratio of
1:0.25 for an allopolyploid with two distinct tetraploid
genomes, as suggested by Lawrence (1970).
Linkage mapping in the K5 population further supported the presence of a predominantly autooctoploid
inheritance as only two out of 1023 markers (0.025%)
were found to be linked in repulsion; all of the remaining
markers were linked in coupling. This observation indicates that although inheritance in dahlia follows a mostly
autooctoploid inheritance with a free recombination of
the eight chromosomes, there is some evidence for a low
frequency of preferential pairing between chromosomes.
This finding is in agreement with other reported cases in
which intermediate forms of inheritance in polyploids
have been observed (Lerceteau-Koehler et al., 2003; Udall
et al., 2005; Stift et al., 2008). Reasons for this preferential
pairing could be that garden dahlias have been shown to
be species hybrids (Lawrence und Scott-Moncrieff, 1935)
and some minor parts of the subgenomes lack sufficient
similarity for free pairing, therefore pairing preferentially. Thus, garden dahlias can also be considered a segmental allooctoploid species hybrid.
Other parameters of the map construction, such
as the level or type of ploidy, are not conclusive. For
each octoploid parent, a total number of eight homologous groups with eight linkage groups each would be
expected. Similarly, eight homologous groups with four
linkage groups each would be expected for tetraploids.
The fact that neither of these numbers matched is due
to the low marker coverage of the Dahlia map. Even the
number of SSR alleles linking the individual linkage
groups into homologous groups was not conclusive, as
the same maximal number of four alleles might also segregate in tetraploid genotypes.
As the lowest chromosome number of the putative
ancestor species of garden dahlias was a chromosome
number of 2n = 32, these ancestors must have already
been polyploids. Furthermore, they were most likely
autopolyploids or at least segmental allotetraploids with
mostly polysomic inheritance because strict allopolyploids with preferential pairing would have resulted in
drastically different inheritance patterns in present-day
garden dahlias. As no Dahlia species with a chromosome number of 2n = 16 are known (Lawrence, 1929), the
diploid ancestors of the polyploid species are most likely
extinct, as suggested by Lawrence (1929).
The genetic architecture of garden dahlias as segmental allooctoploids with mostly octosomic inheritance (all eight chromosomes segregate randomly and
no preferential pairing occurs) is in agreement with our
observation that a large number of morphological traits
do not show segregation into distinct classes but instead
vary continuously among the members of the biparental
mapping population (data not shown). Therefore, simple
selection schemes for major dominant traits, such as disease resistance, in variety breeding will not be very efficient. Furthermore, this mode of inheritance also makes
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it difficult to detect molecular markers linked to traits of
interest, and therefore impedes marker-assisted selection
in dahlias. An alternative would be the gradual enrichment of favorable alleles in breeding stocks to increase
the frequency of hybrids with higher allele dosages of
these favorable alleles.
In summary, our results show that molecular markers are useful tools for genome analysis in complex
polyploid, nonmodel plant genomes, even without dense
marker maps and extensive sequence information.
Acknowledgments
The authors thank Professor Michael Otto from
Lueneburg for the financial support of the project.
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